Methods for producing hydroxy amino acids, esters, and derivatives thereof

ABSTRACT

A method for producing a hydroxy-amino ester, or derivative thereof, is provided. A substituted β-ketodiester having a ketone group and two ester functional groups is contacted with a ketoreductase under conditions permitting the reduction of the ketone group to an alcohol. Only one of the ester functional groups is regioselectively hydrolyzed to the corresponding carboxylic acid, whereby a non-hydrolyzed ester functional group remains. The carboxylic acid is converted to an amine or a derivative thereof to produce a hydroxy-amino ester or derivative thereof. A number of novel hydroxy-amino esters are prepared by the method.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part, and claims priority, of U.S.application Ser. No. 10/237,831, filed Sep. 9, 2002, the entire contentsof which are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to methods for the production of chiralcompounds, and in particular to methods for the production of chiralhydroxy-amino compounds. The hydroxy-amino compounds have applicationsin the synthesis of pharmaceutical products.

BACKGROUND

Natural and non-natural α-hydroxy-β-amino acids and β-hydroxy-γ-aminoacids and their derivatives occur in many biologically active naturalproducts and are important intermediates in the synthesis of variouspharmaceuticals. One of the most important α-hydroxy-β-amino acids isthe side chain of the potent anticancer drug Taxol. Various derivativesof this β-amino acid have been synthesized and linked to the polycycliccore ring of Taxol in an effort to improve the potency and the spectrumof uses of this important drug.

The β-hydroxy-γ-amino acid structural motif is encountered in a numberof natural products and current and developmental drugs. Some of themost common β-hydroxy-γ-amino acids include statine, isostatine andbenzyl statine (phenylstatine) (FIG. 1). Statine is the key component ofpepstatin, a naturally-occurring hexapeptide antibiotic, which acts asan inhibitor of aspartic acid proteases such as rennin, pepsin andcathepsin D [Umezawa, H etal J. Antibiotics 23, 259 (1970); Ric, D. H.J. Med. Chem 23, 27 (1980)]. The low selectivity of pepstatine has ledto the development of more specific synthetic analogues by substitutingthe isobutyl moiety of statine with more lipophilic substituents such ascyclohexylmethyl, which led to the widely used analoguecyclohexyl-statine. Isostatine is an essential amino acid in Didemnins[Sakai, R. at al J. Am. Chem. Soc. 117, 3734 (1995); Joullie, M. M. J.Am. Chem. Soc 112, 7659 (1990)], a group of cyclic peptides which showstrong antitumor, antiviral, and immunosuppressive activity (Sakai, R.et al. J. Med. Chem. 39, 2819 (1996)]. Benzyl statine is part of thebiologically active compounds hapalosin (Stratmann, K et al J. Org.Chem. 59, 7219 (1994); Armstrong, R. W. J. Org. Chem. 60, 8118, (1995)]and dolastatin 10 [Shiori, T et al Tetrahedron 49, 1913 (1993)]. Inparticular hapalosin restores the lethal activity of cytotoxic antitumordrugs (such as actinomycin D, colchicines and taxol) to cancer cells bybreaking the P-glycoprotein-mediated multi-drug resistance caused by theexport of the cancer drugs from the cell using transmembraneP-glycoproteins.

Other α-hydroxy-γ-amino acids that are incorporated in molecules withbiological activities include (2S,3S,4R)-4-amino-3-hydroxy-2-methylpentanoic acid, which is the amino acid linker of bleomycin B2 and themain constituent of the powerful carcinostatic blenoxane [Boger, D. L etal J. Am Chem Soc 116, 5607, (1994)] and(2R,3S,4R)-4-amino-3-hydroxy-2-methyl-5-(2′-pyridil) pentanoic acid,which is part of pyridomycin [Kinoshita, M; Awamura, M. Bull. Chem. Soc51, 869 (1978)], a Streptomyces-synthesized anti-mycobacterial drug(FIG. 2). Statines and related compounds based on β-hydroxy-γ-aminoacids are particularly prevalent in anti-cancer drugs and drugcandidates. The absolute stereochemistry of these molecules is importantfor biological activity.

Another important motif in pharmaceutically-active compounds is theα-hydroxy-β-amino acid structural unit. Among the examples ofpharmaceutical products that contain the α-hydroxy-β-amino acid moietyas a key component in their structures are molecules such as bestatin,amastatin and ubenimex, which possess immunoregulatory, antitumor andantimicrobial activities. The ability to prepare compounds in this classwith defined absolute stereochemistry is critical to the commercialsynthesis of these compounds and their analogs.

Despite the general importance of hydroxyl-substituted β- and γ-aminoacids and their derivatives as pharmaceutical intermediates, thepreparation of these compounds remains a significant challenge tochemists. Most of the synthetic approaches toward the production ofα-hydroxy-β-amino acids are purely chemical transformations that requiremulti-step reaction sequences, chiral catalysts or starting materials,and stringent or air-sensitive reaction conditions. Occasionally thesynthetic methods involve the production of relatively unstableintermediates. Most of the chemical syntheses of statine and isostatine,for example, begin from the natural α-amino acids leucine andisoleucine, respectively [Hamada, Y. et al J. Am Chem Soc, 111, 669(1989); Tao, J.; Hoffmann, R. V J. Org. Chem 62, 2292 (1997)]. Afterprotection of the amino group (PG=protecting group), an aldol or Claisencondensation to the β-keto-γ-amino acid followed by a reduction givesthe desired β-hydroxy γ-amino acid product (FIG. 3).

Some of the problems encountered in these syntheses are theisomerization of the γ-carbon under the basic conditions of thecondensation reaction, the many steps required (often 7-10), and the lowdiastereoselectivity of the final reduction step, which often timesgives the wrong diastereomer as the major product [Kessler, H; Schudok,M Synthesis 457 (1990); Maibaum, J.; Rich, D. H J. Org. Chem 53, 869(1988)]. An obvious drawback in using methods based on natural aminoacid precursors for the synthesis of β-hydroxy-γ-amino acids is thatnon-natural α-amino acid counterparts cannot always be easily accessed,and for this reason other chemical synthetic schemes have beendeveloped. The β,γ-amino alcohol moiety in one alternative syntheticroute is synthesized from α,β-unsaturated alcohols that are epoxidizedusing a chiral catalyst, followed by a ring opening using an nitrogennucleophile (FIG. 3) [Catasus, M. et al Tetrahedron Lett 40, 9309(1999); Catejon, P. et al Tetrahedron 52, 7063. (1996)]. Although goodenantiomeric purity of the product was reported (90-99% ee), thismethodology is long (6-10 steps), gives moderate yields (20-40%), andrequires expensive catalysts and stringent air-sensitive reactionconditions. Other methods for synthesizing β-hydroxy-γ-amino acidsinvolve Wittig reactions of chiral oxazolidinones [Reddy, G. V et alTetrahedron Lett 40, 775 (1999)] asymmetric Claisen rearrangements[Krebs, A.; Kazmaier, U. Tetrahedron Lett. 40, 479 (1999)], selectiveGrignard reaction of N-protected amino acids [Veeresha, G.; Datta, ATetrahedron Lett 38, 5223 (1997)] or the use of doubly chiral precursors[Kwon and Ko, Tetrahedron Lett 43, 639-641 (2002)]. Again, long andcomplicated reaction sequences and chiral starting materials and/orcatalysts are required using these methodologies.

Enzyme catalysis offers an alternative to purely chemical syntheticschemes. Enzymatic methods that have been reported to date areresolutions of a racemic mixture, having a maximum yield of 50% for theresolution step alone. Challenges similar to those encountered in thechemical synthesis of β-hydroxy-γ-amino acids are also faced in thechemical synthesis of α-hydroxy-β-amino acids. In both cases, gainingcontrol over the stereochemistry of the chiral carbons bearing both theamino and the alcohol groups at reasonable cost and high enantiomericpurity is the key to the successful production of these importantchemical intermediates.

Chiral hydroxy compounds can be produced by the stereoselectivereduction of ketones catalyzed by ketoreductase enzymes. As used herein,the term ketoreductase means any enzyme that catalyzes the reduction ofa ketone to form the corresponding alcohol. Ketoreductase enzymesinclude those classified under the Enzyme Commission numbers of 1.1.1.Such enzymes are given various names in addition to ketoreductase,including, but not limited, to alcohol dehydrogenase, carbonylreductase, lactate dehydrogenase, hydroxyacid dehydrogenase,hydroxyisocaproate dehydrogenase, β-hydroxybutyrate dehydrogenase,steroid dehydrogenase, sorbitol dehydrogenase, aldoreductase, and thelike.

Many examples of enzymatic reductions of various classes of substrateshave been reported [Wong, C-H; Whitsides, G. M. Enzymes in SyntheticOrganic Chemistry, Pergamon, N.Y., (1994); Sugai, T Curr. Org. Chem 3,373 (1999)]. Various alcohol dehydrogenases have been investigated[Patel, R. N Adv. Appl. Microbiol 43, 91 (1997); Riva, S.; Carrea, G.Angew. Chem. Int. Ed 39, 2226 (2000)]. A well known example is horseliver alcohol dehydrogenase (HLADH), an enzyme that has been veryextensively studied and can reduce aldehydes and ketones to thecorresponding alcohols, in some cases providing alcohols in goodenantiomeric purity. The substrate range is limited and does not includemost β-ketoesters, however.

Various ketoreductase enzymes have been identified that catalyze thestereoselective reduction of a range of different ketones, includingβ-ketoesters. [See, for example, J. David Rozzell, ACS Symposium Series776, Applied Biocatalysis in Specialty Chemicals and Pharmaceuticals, B.C. Saha and D. C. Demirjian, eds., pp.191-199, (2000) and referencestherein, all hereby incorporated by reference.] These enzymes have beenshown to act on a number of structurally diverse ketones. The genesexpressing a number of these broad-range ketoreductases have been clonedand expressed, and a number of these enzymes are readily availablecommercially (BioCatalytics, Inc, Pasadena, Calif. USA). In many cases,enzymes can be identified that can produce either stereoisomer of achiral alcohol by stereoselective reduction of a target ketone. Forexample, when the Ketoreductase Screening Set (Catalog number KRED-8000,BioCatalytics, Inc, Pasadena, Calif. USA) containing 8 differentketoreductases was screened against either alpha-chloroacetophenone orethyl 4-chloroacetoacetate, some enzymes could be found within the setthat were R-selective while others were found that were S-selective withrespect to the chiral alcohol produced.

It has also been demonstrated that ketoreductase enzymes can be used tocatalyze the reduction of 2-substituted-3-ketoesters. The products ofthese reductions are compounds with two chiral centers, and depending onthe enzyme employed, the reduction can be diastereoselective, as shownin FIG. 4. Such reactions have been described using isolated enzymes andwith whole cells. When the enzymes within the Ketoreductase ScreeningSet (Catalog number KRED-8000, BioCatalytics, Inc, Pasadena, Calif. USA)were studied for the reduction of 2-ethyl-3-ketobutyrate ethyl ester,certain enzymes were shown to be highly diastereoselective for thereduction to the corresponding alcohol. [For other examples, see S.Rodriguez et al., J. Org. Chem., 65, 2586 (2000); S. Rodriguez et al.,J. Am. Chem. Soc., 123, 1547 (2001) and references therein, herebyincorporated by reference.]

Benner and coworkers used actively fermenting Baker's yeast to carry outthe reduction of the compounds shown in FIG. 5 where n is 1 and R isallyl or propargyl [T. Arsian and S. A. Benner, J. Org. Chem., 58,2260-2264 (1993) and references therein, hereby incorporated byreference]. These compounds were prepared as potential precursors forthe synthesis of non-standard nucleic acid bases. These were the onlycompounds for which reduction with fermenting yeast was reported, andthe ketoreductase or ketoreductases involved were neither isolated nordetermined. The reduction reaction was reported to be enantioselectiveand diastereoselective, although the degree of selectivity observedvaried widely depending on reaction conditions, and yields in some caseswere diminished by the partial metabolism of the substrate.

There are no reports of the highly diastereoselective reduction of arange of substituted β-ketodiesters, nor any reports of the use ofsubstituted β-ketodiesters in the production of α-hydroxy-β-amino acidsand β-hydroxy-γ-amino acids using a reaction sequence incorporating adiasereoselective reduction of substituted β-ketodiesters.

DESCRIPTION OF THE INVENTION

The present invention is directed toward methods for the production ofhydroxy-amino acids, esters, and derivatives, includingα-hydroxy-β-amino acids, esters, and derivatives and β-hydroxy-γ-aminoacids, esters, and derivatives. The methods of the present invention arebroadly applicable for the synthesis of a wide range of chiral hydroxyβ- and γ-amino acids from inexpensive and easily accessible startingmaterials.

In one embodiment, the invention is directed to a method for producing ahydroxy-amino acid, ester, or derivative thereof. A substitutedβ-ketodiester having a ketone group and two ester functional groups iscontacted with a ketoreductase under conditions permitting the reductionof the ketone group to an alcohol. Only one of the ester functionalgroups is regioselectively hydrolyzed to the corresponding carboxylicacid, whereby a non-hydrolyzed ester functional group remains. Eitherthe carboxylic acid or the non-hydrolyzed ester functional group isconverted to an amine or a derivative thereof to produce a hydroxy-aminoacid, ester, or derivative thereof.

A key step in the preparation of the target compounds is thediastereoselective reduction of substituted β-ketodiesters to form thecorresponding substituted hydroxydiesters. In one embodiment, the methodof the present invention uses a reaction sequence comprising adiastereoselective enzyme-catalyzed reduction of a β-ketodiester tointroduce two or more chiral centers in a single step, followed byregioselective hydrolysis of only one of the two ester functional groupsto form the corresponding carboxylic acid, and a conversion including astereospecific chemical rearrangement in which the carboxylic acid isconverted to an amine, or derivative thereof, to generate the desiredhydroxy-amino ester, or derivative thereof. In another embodiment, themethod of the present invention uses a reaction sequence comprising adiastereoselective enzyme-catalyzed reduction of a β-ketodiester,followed by regioselective hydrolysis of only one of the two esterfunctional groups to form the corresponding carboxylic acid, theconversion of the non-hydrolyzed ester functional group to an amide, ahydrazide, or a hydroxamic acid derivative, and a stereospecificchemical rearrangement in which the amide, hydrazide, or hydroxamic acidderivative is converted to an amine, or derivative thereof, to generatethe desired hydroxy-amino acid or derivative thereof. As used herein, asit pertains to a hydroxyamino compound, the words “derivative thereof”means a carbamate or urethane, which can be cyclic or acyclic, a urea, ahydrazide, or an amide formed from the amino group, or any protectedform of the alcohol, including ethers, silyl ethers, alkyl esters, arylesters, aralkyl esters, or carbonate esters.

In connection with this invention, it has been discovered that, whencontacted with an appropriate ketoreductase, a broad range ofsubstituted β-ketodiesters, such as 3-substituted-oxaloacetic diesters(n=0 in FIG. 5) and 2-substituted-3-ketoglutarate diesters (n=1 in FIG.5), can be reduced diastereoselectively as shown in FIG. 5, producing asubstituted β-hydroxydiester with 2 chiral centers. Preferably, thereaction catalyzed by the ketoreductase is substantiallydiastereoselective. As used herein, the term “substantiallydiastereoselective” means a reaction that produces a compound containingtwo or more chiral centers with the product mixture containing at leastabout 50% of a single diastereomer of the possible diastereoisomers,preferably at least about 75% of a single diastereomer, and morepreferably at least about 90% of a single diastereomer. As a chiralsynthesis rather than a resolution, yields up to 100% of theoretical canbe achieved during the enzymatic reductions, with two chiral centersbeing introduced in a single step in a substantially diastereoselectivemanner. The diastereomeric hydroxy diesters can be further converted toα-hydroxy-β-amino acids and β-hydroxy-γ-amino acids by regioselectivehydrolysis of only one of the two ester functional groups and conversionof the remaining ester or carboxylic acid to an amine by astereospecific rearrangement reaction which preserves the asymmetry atboth chiral centers. In a particularly preferred embodiment, theβ-ketodiester is a compound having the following formula:

wherein n is 0 or 1, and R¹, R² and R³ are each independently selectedfrom the group consisting of alkyl, substituted alkyl, aryl, substitutedaryl, aralkyl, substituted aralkyl, heterocyclic, and substitutedheterocyclic.

It will be appreciated that, when n is 0, a diester having the aboveformula can be denoted as an α-ketodiester (relative to the right end ofthe formula), as well as a β-ketodiester (relative to the left end ofthe formula). And, indeed, the conversion of α-ketodiesters as well asβ-ketodiesters is encompassed by the present invention.

As used herein, the term “alkyl,” alone or in combination, means astraight-chain or branched-chain hydrocarbon, either saturated orunsaturated, containing from 1 to about 12 carbon atoms. “Substitutedalkyl” means alkyl groups substituted on one or more carbon atoms withone or more substituents selected from the group consisting of hydroxy,alkoxy, thio, thioalkyl, fluoro, chloro, bromo, iodo, carboxy,carboxyalkyl, carbamoyl, carbamide, amino, amidino, phosphate,phosphonate, phosphinate, phosphinyl, their derivatives, and the like.

As used herein, the term “aryl,” alone or in combination, means acarbocyclic aromatic system containing from 1 to 4 rings, wherein saidrings may be attached in a pendant manner to each other or may be fusedto each other. Examples of aryl groups include phenyl, naphthyl,biphenyl, anthracenyl, and the like. Substituted aryl means aryl groupssubstituted on one or more carbon atoms with one or more substituentsselected from the group consisting of hydroxy, alkoxy, thio, thioalkyl,fluoro, chloro, bromo, iodo, carboxy, carboxyalkyl, carbamoyl,carbamide, amino, amidino, phosphate, phosphonate, phosphinate,phosphinyl, their derivatives, and the like.

As used herein, the term “aralkyl” means an alkyl group as defined abovesubstituted with an aryl group as defined above. Substituted aralkylmeans aralkyl groups substituted on one or more carbon atoms with one ormore substituents selected from the group consisting of hydroxy, alkoxy,thio, thioalkyl, fluoro, chloro, bromo, iodo, carboxy, carboxyalkyl,carbamoyl, carbamide, amino, amidino, phosphate, phosphonate,phosphinate, phosphinyl, their derivatives, and the like.

As used herein, the term “heterocyclic,” alone or in combination, meansa saturated or unsaturated monocyclic or multi-cyclic group containingone or more heteroatoms selected from the group consisting of oxygen,nitrogen, sulfur, phosphorous, selenium, and silicon. Substitutedheterocyclic means heterocyclic groups substituted on one or more carbonatoms with one or more substituents selected from the group consistingof hydroxy, alkoxy, thio, thioalkyl, fluoro, chloro, bromo, iodo,carboxy, carboxyalkyl, carbamoyl, carbamide, amino, amidino, phosphate,phosphonate, phosphinate, phosphinyl, their derivatives, and the like.

Some ketoreductase enzymes particularly useful in the present inventionrequire the presence of nicotinamide cofactors in order to catalyze thereduction of the subject β-ketodiesters. As used herein, the term“nicotinamide cofactors” includes NAD+, NADH, NADP+, NADPH, and anyderivatives thereof that can be used as cofactors by oxidoreductaseenzymes. Nicotinamide cofactors useful in the present invention arereadily available commercially from vendors, including Sigma-AldrichChemical Company (St. Louis, Mo. USA), BioCatalytics, Inc., (Pasadena,Calif. USA), Roche Diagnostics (Indianapolis, Ind. USA) and others wellknown to those skilled in the art. Derivatives of nicotinamide cofactorsuseful in the practice of this invention include the nicotinamideanalogs reported in U.S. Pat. No. 5,801,006, the disclosure of which ishereby incorporated by reference, polyethyleneglycol functionalizednicotinamide molecules such as reported by Okada and Urabe in Methods inEnzymology, 136, 34-45 (1987), and the like. The concentration ofnicotinamide cofactor used in the reaction mixture with a ketoreductaseenzyme preferably ranges from about 0.001 mM to about 10 mM, and morepreferably from about 0.01 mM to about 0.5 mM. For the stereoselectivereduction to be carried out as described in the present invention, thereduced form of the nicotinamide cofactor (NADH, NADPH or analogthereof) is used by the ketoreductase enzyme. It is also possible tostart with an oxidized form of the nicotinamide cofactor (NAD+, NADP+,or analog thereof), which is less expensive that the reduced form,provided that a source of reducing equivalents is furnished to reducethe oxidized form of said cofactor to the reduced form for theenzyme-catalyzed reduction to proceed.

In the method of the present invention, the nicotinamide cofactors canbe recycled, if desired. Cofactor recycling can be achieved in cell-freeenzymatic reactions by the use of an appropriate recycling enzyme incombination with a ketoreductase. Enzymes useful for the recycling ofnicotinamide cofactors are well-known in the art, and include formatedehydrogenases, glucose dehydrogenases, sorbitol dehydrogenases, alcoholdehydrogenases and the like. Any of the recycling methods known in theart may be used in the practice of this invention. Some examples ofcofactor recycling methods are described in PreparativeBiotransformations (S. M. Roberts, editor), 3.1.1-3.1.6, John Wiley &Sons, Chichester, U.K. (1996) and references therein; Z. Shaked and G.M. Whitesides, J. Am. Chem. Soc. 102, 7104-5 (1980) and referencestherein; J. B. Jones and T. Takamura, Can. J. Chem. 62 77 (1984); allhereby incorporated by reference.

In the practice of this invention, cofactor recycling may also beachieved by the use of a microorganism into which the genes encodingboth the ketoreductase and the recycling enzyme have been cloned andexpressed together. In this embodiment, the whole cell may be used asthe catalyst, or, if desired, the ketoreductase and the recyclingenzymes may be isolated from the cell. As used herein with respect toenzymes, the term “isolated” means extracted from or separated fromcells. An isolated enzyme or enzymes may be used as a crude cell lysate,partially purified enzyme preparation, or a purified enzyme preparation.

In accordance with this invention, the ketoreductase and the recyclingenzyme may be used as soluble enzymes or, if desired, one or bothenzymes may be immobilized prior to use. When used as soluble enzymes,ketoreductases and recycling enzymes useful in the practice of thisinvention may be isolated from cells capable of producing the desiredenzymes and used without purification, or purified partially orcompletely. The purification of the enzymes may be accomplished bytechniques well known to those skilled in the art. Some examples ofpurification methods for enzymes are described in Methods in Enzymology,22 (1971) and references therein, hereby incorporated by reference.

If the ketoreductase and the recycling enzymes are to be immobilized,techniques well known in the art can be used. Either the ketoreductaseenzyme or the cofactor recycling enzyme may be immobilized separately,or both enzymes may be immobilized together. Such immobilization of theenzymes can be carried out by co-immobilization of both enzymes togetheron the same support material, or the ketoreductase and the recyclingenzyme may be immobilized separately and the two immobilized enzymes canbe combined in appropriate amounts for carrying out thediastereoselective reduction reaction. The appropriate amounts ofimmobilized enzymes to be used can be readily determined by personsskilled in the art. Methods for the immobilization of enzymes are wellknown to those skilled in the art. One example of an immobilized enzymemethod useful in the practice of this invention is described by Weetallet al., Methods in Enzymology 34, 59-72 (1974), which is herebyincorporated by reference. In this method, enzymes may be immobilized onan amine-functionalized porous glass or ceramic support which has beenactivated with glutaraldehyde. It is also possible that whole cellscontaining the ketoredeuctase enzyme or both the ketoreductase enzymeand a recycling system may be immobilized, if desired, in the practiceof this invention. Various exemplary methods for immobilization of bothwhole cells and enzymes which may be used in the practice of thisinvention are described in Methods in Enzymology 44 (1976), K. Mosbacheditor, Immobilization of Enzymes and Cells, Gordon F. Bickerstaff, ed.,Humana Press, Totowa, N.J. (1997) and in Biocatalytic Production ofAmino Acids and Derivatives, D. Rozzell and F. Wagner, Eds., HanserPublishers, Munich, (1992) pp. 279-319, all hereby incorporated byreference. It is understood that other similar methods exist and mayalso be used in the practice of this invention.

In the next step of the method of this invention, the substitutedβ-hydroxydiester, which is the immediate product of thediasereoselective reduction of the substituted β-ketodiester, ishydrolyzed regioselectively to the mono-carboxylic acid (FIG. 6).

As used herein, the term “hydrolyzed regioselectively” refers to theconversion of only one of the two ester groups (either the esteradjacent to the R-group or the ester 2 carbons removed from the R-group)as shown in the structure in FIG. 6 to the substantial exclusion of theother. As it relates to hydrolyzing regioselectively an ester in amolecule containing two or more ester groups, the term “substantialexclusion” means converting at least about 80%, preferably at leastabout 90%, and more preferably at least about 95% of one ester groupwhile converting less than about 20%, preferably less than about 10%,and more preferably less than about 5% of any other ester group in themolecule. As long as only one of the two ester functional groups ishydrolyzed regioselectively to the substantial exclusion of the other,regardless of which one is hydrolyzed, the two carboxylate groups becomechemically distinguishable as the non-hydrolyzed ester and thecarboxylic acid.

Regioselective hydrolysis can be achieved enzymatically using ahydrolytic enzyme. Any hydrolytic enzyme capable of regioselectivehydrolysis of the substituted β-hydroxydiester to the mono-carboxylicacid may be used. Suitable enzymes for this regioselective hydrolysisinclude proteases, amidases, lipases, esterases and the like. Many broadrange lipases, proteases, and esterases are known that can hydrolyzeesters with high regioselectivity. Suitable enzymes for regioselectivehydrolysis of a given substituted β-hydroxydiester in accordance withthe invention can be identified by routine screening of varioushydrolytic enzymes. Examples of such hydrolytic enzymes can be found inthe Chirazyme Screening Set or the ICR Screening Set, both availablefrom BioCatalytics Inc. (Pasadena, Calif. USA). In a typical screeningexperiment, individual reaction mixtures are set up with each of thecandidate hydrolytic enzymes and the target substituted β-hydroxydiesterto be regioselectively hydrolyzed, and the progress of the reaction ismonitored by any convenient assay method. Such assay methods include,but are not limited to, gas chromatography, thin-layer chromatography,high performance liquid chromatography, and the like. It is well knownby persons skilled in the art how to identify and select a suitablehydrolytic enzyme for regioselective hydrolysis.

Alternatively, regioselective hydrolysis of the substitutedβ-hydroxydiester can be accomplished chemically by using an appropriatebase under reaction conditions permitting the regioselective hydrolysisof only one of the two ester functional groups to the substnatialexclusion of the other. Such regioselective hydrolysis can be achievedusing a variety of different bases, including, but not limited to,mineral bases such as sodium hydroxide, potassium hydroxide, calciumhydroxide, barium hydroxide, sodium carbonate, potassium carbonate, andcalcium carbonate. Ammonium hydroxide may also be used. Other bases thatmay be used for regioselective hydrolysis in the practice of thisinvention include tertiary amine bases such as triethylamine,trimethylamine, tributylamine, tribenzylamine, and the like. Theregioselective hydrolysis may be carried out in an aqueous reactionmedium, in an aqueous reaction medium containing various amounts ofadded organic solvent, or in an organic medium containing only smallamounts of water. The reaction medium may also be an aqueous/organictwo-phase system, if desired. Determination of appropriate conditionsfor achieving regioselective mono-hydrolysis can be accomplished byroutine experimentation by those skilled in the art. It the practice ofthis invention, it has been found that too high a molar ratio ofbase:substituted β-hydroxydiester results in over-hydrolysis (that is,hydrolysis of both of one ester functional groups, or at least some ofthe second ester functional group); whereas too low a molar ratio ofbase:substituted β-hydroxydiester results in residual amounts ofunhydrolyzed diester. For achieving regioselective mono-hydrolysis ofthe substituted β-hydroxydiester, the preferred molar ratio ofbase:substituted β-hydroxydiester ranges from about 1:1 to about 1.5:1.The preferred temperature range for the hydrolysis reaction is fromabout 4° C. to about 100° C., and more preferably from about 20° C. toabout 50° C. Although aqueous reaction conditions are typically employedfor the regioselective mono-hydrolysis of the substitutedβ-hydroxydiester, co-solvents may be used, if desired, to improve thesolubility of the diester or to modulate the rate of the reaction.Suitable co-solvents include ethanol, methanol, isopropanol,tetrahydrofuran, dioxane, dimethylsulfoxide, dimethyl formamide, and thelike.

Following the regioselective mono-hydrolysis of the substitutedβ-hydroxydiester, either the carboxylic acid or the ester functionalgroup is converted to an amine, or derivative thereof, by means of astereospecific chemical rearrangement. The rearrangement reactions thatcan be used in the practice of this invention include the Curtiusrearrangement and modified versions of the Curtius rearrangement, theLossen rearrangement and the Hoffmann rearrangement (FIG. 6). Theserearrangement reactions are well-studied reactions that are well knownto those skilled in the art.

In one embodiment, the carboxylic acid is converted to an amine, orderivative thereof, by means of the Curtius-type rearrangement using thereagent diphenylphosphoryl azide (DPPA). Heating of the mono-carboxylicacid, produced by regioselective mono-hydrolysis of the substitutedβ-hydroxydiester, with DPPA in the presence of stoichiometric amounts oftriethyl amine (TEA) in toluene gives in one step the aminoalcoholrearrangement product as the cyclic carbamate (urethane) derivative(FIG. 7).

Formation of the mono acid to the corresponding acid azide aftertreatment with oxalyl chloride and sodium azide, and rearrangement byheating of the azide in tert-butanol or ethanol gave the cycliccarbamate (FIG. 7) as the major product. Small amounts of the ethylcarbamate can be observed when the reaction is carried out in ethanol.The urethane derivatives can be further converted to the correspondingamines, if desired, by hydrolysis under either acidic or basicconditions. The hydroxy group may be protected, if desired, prior torearrangement. Typical protecting groups include, but are not limitedto, simple esters such as acetyl, butyryl, benzoyl, phenylacetyl, andthe like; ethers such as methyl ethoxymethyl, dihydropyranyl, and thelike; silyl ethers such as t-butyl dimethyl silyl, trimethyl silyl, andthe like; and carbonate esters such as t-butyloxycarbonyl,carbobenzyloxy, and the like.

In another embodiment of this invention, the non-hydrolyzed esterfunctional group is converted to an amine. This conversion isaccomplished in two steps. First, the ester is reacted with ammonia,hydrazine, or hydroxylamine to form the corresponding carboxamide,hydrazide, or hydroxamic acid, respectively. Then, the carboxamide,hydrazide, or hydroxamic acid is subjected to conditions permitting thestereospecific rearrangement to form the amine, or a derivative thereof.In each case, the rearrangement proceeds with retention of configurationof the migrating atom, and as a result, the optical purity of the finalproduct is dependent on the stereoselectivity of the enzymaticreduction.

The stereospecific rearrangement may be carried out on the carboxamidevia the Hofmann-type rearrangement [E. S. Wallis and J. F. Lane, OrganicReactions III, 267 (1949) and references therein; P. A. S. Smith, Trans.N.Y. Acad. Sci. 31, 504 (1969) and references therein; S. Simons, J. OrgChem. 38, 414 91973) and references therein; W. L. F. Armarego et al, J.Chem. Soc. Perkin Trans. I, 2229 (1976) and references therein; allhereby incorporated by reference]; on the hydroxamic acid via the Lossenrearrangement [S. Bittner et al (Tet. Lett. 23, 1965-8 (1974) andreferences therein; L. Bauer and O. Exner, Angew. Chem. Int. Ed. 13, 376(1974) and references therein; all hereby incorporated by reference]; oron the hydrazide via the Curtius rearrangement [Yamada, S. Chem. Pharm.Bull. 22, 1398 (1974); P. A. S. Smith, Organic Reactions III, 337 (1946)and references therein; J. H. Saunders and R. J. Slocombe, Chem. Rev.43, 205 (1948) and references therein; D. V. Banthorpe in The Chemistryof the Azido Group, S. Patai Ed., Interscience, New York, 1971, pp.397-405 and references therein; J. D. Warren and J. D. Press, Synth.Comm. 10, 107 (1980) and references therein; all hereby incorporated byreference].

The invention will now be further described by the following examples,which are presented here for illustrative purposes only and are notintended to limit the scope of the invention.

EXAMPLES Example 1 Synthesis of 2-benzyl-3-ketoglutarate diethyl ester

To 100 mL of dry acetone was added diethyl 1,3 acetone dicarboxylate (20mL, 0.11 mole), benzyl bromide (8 mL, 0.067 mole) and potassiumcarbonate (18 grams, 0.13 mole). The heterogeneous mixture was refluxedovernight with vigorous stirring. After 16 hours the reaction progresswas checked by thin layer chromatography. All the benzyl bromidestarting material had been consumed and an intense spot corresponding tothe product 2-benzyl-3-ketoglutarate diethyl ester was observed. Thesolution was filtered, and the solids were washed with another 100 mL ofacetone. The combined organic solutions were evaporated to dryness,leaving a yellow liquid. After silica gel chromatography using ethylacetate/hexane (2/8, v/v) as the elution solvent, the product wasobtained as a colorless liquid (17.4 grams, 89% yield). The actual yieldtowards the formation of 2-benzyl-3-ketoglutarate diethyl ester waslower than 89% since a byproduct (probably the benzyl ether forming fromoxygen substitution) coelutes from the silica column along with the2-benzyl substituted product. For an alternative synthetic method, seeExample 7.

Example 2 Synthesis of 2-methyl-3-ketoglutarate diethyl ester

To 20 mL of dry tetrahydrofuran that had been purged with nitrogen wasadded 5 mL of diethyl 1,3 acetone dicarboxylate (27 millimole), and thesolution was cooled to −15° C. prior to the dropwise addition of 15 mLof 2 M lithium diisopropyl amide (30 millimole). The reaction mixturewas maintained under a nitrogen atmosphere at −15° C., and 3 mL (48millimole) of iodomethane were added slowly to the reaction mixture. Thereaction was allowed to reach room temperature gradually overapproximately 2.5 hours, and stirring was continued overnight. After 16hours, the reaction mixture was poured into 150 mL of a 1:1 mixture of0.1 N aqueous HCl and diethyl ether. The organic and aqueous layers wereseparated, the aqueous layer was extracted two more times with diethylether, and the combined organic extracts were back extracted with brine,dried over anhydrous Na₂SO₄ and evaporated to dryness. A yellow liquidwas obtained which on gas chromatographic analysis showed a compositionconsisting of 47% 2-methyl-3-ketoglutarate diethyl ester and 20% of thedisubstituted product 2,4-dimethyl-3-ketoglutarate diethyl ester. Afterpurification by silica gel chromatography using ethyl acetate/hexane(2/8, v/v) as the elution solvent, 1.5 grams of purified2-methyl-3-ketoglutarate diethyl ester was obtained as a colorlessliquid. The isolated yield was 30%.

Example 3 Synthesis of 2-isobutyl-3-ketoglutarate diethyl ester

Isobutyl iodide (2.5 mL, 14 millimole), diethyl-1,3-acetonedicarboxylate (2.5 mL 14 millimole) and powdered K₂CO₃ (2.5 grams, 18millimole) were combined in 15 mL of dry acetone, and the heterogeneousmixture was refluxed overnight. After 16 hours, the reaction mixture wasfiltered through diatomaceous earth (Celite), the solids were washedwith another 30 mL of dry acetone, and the combined organic solutionswere evaporated to dryness. The product 2-isobutyl-3-ketoglutaratediethyl ester was further purified by silica gel chromatography (ethylacetate/hexane, v/v, 2/8), providing 2-isobutyl-3-ketoglutarate diethylester as a colorless oil (1 gram, 40% isolated yield).

Example 4 Screening of Enzymes to Determine the Best Ketoreductase forReduction of 2-methyl-3-ketoglutarate diethyl ester

The Ketoreductase Screening Set (KRED-8000; BioCatalytics, Inc.,Pasadena, Calif. USA) containing eight different ketoreductases wasscreened to determine the best enzyme for the diastereoseelctivereduction of 2-methyl-3-ketoglutarate diethyl ester. Eight individualreaction mixtures were set up containing, in 1 mL of 300 mM potassiumphosphate pH=6.5, the following components: 25 mM2-methyl-3-ketoglutarate diethyl ester, 5 mM NADPH, 100 mM NaCl, 10%(v/v) glycerol, 200 mM glucose, 5% (v/v) DMSO, and 2 mg glucosedehydrogenase for NADPH recycling. To each reaction mixture was added 5milligrams of a different lyophilized ketoreductase KRED 1001. All eightreactions were left at 37° C. overnight. After 16 hours, each reactionmixture was extracted with ethyl acetate and the reaction products wereanalyzed using gas chromatography. The enzymes in the screen and theyield of reduction product obtained were as follows: KRED 1001, 97%;KRED 1008, 90%; KRED 1007, 55%; KRED 1004, 59%. The yield of reductionproduct was 10% or less for the other 4 KRED enzymes.

Example 5 Screening of Enzymes to Determine the Best Ketoreductase forReduction of Other 2-Substituted-β-ketodiesters

The method of Example 4 was repeated except that2-methyl-3-ketoglutarate diethyl ester was replaced with either2-benzyl-3-ketoglutarate diethyl ester or 2-isobutyl-3-ketoglutaratediethyl ester. The best enzyme for the reduction of2-benzyl-3-ketoglutarate diethyl ester was KRED 1008, and the bestenzymes for the reduction of 2-isobutyl-3-ketoglutarate diethyl esterwere KRED 1008 and KRED 1001.

Example 6 Chiral Analysis of the Diastereoselectivity of the Reductionof 2-Substituted-β-ketodiesters

Analysis of the diastereoselectivity of the reduction of2-substituted-β-ketodiester was performed. The enzymatic reductionproducts of 2-methyl-3-ketoglutarate, 2-benzyl-3-ketoglutarate, and2-isobutyl-3-ketoglutarate were analyzed using chiral gas chromatographyusing a ChiralDex column (Chiral Technologies). For the2-methyl-3-hydroxyglutarate diester, the analysis was carried out underisocratic conditions at 130° C. In the case of isobutyl and benzylsubstituted compounds, successful separation of all four diastereomerswas achieved after derivatizing the reduction products as thecorresponding acetate esters by reaction with acetic anhydride and usinga temperature gradient. A typical protocol for the preparation of thesecompounds in small scale was as follows: a sample (5-10 mg) of alcoholwas dissolved in 0.5 mL diethyl ether, and 1 drop of triethylamine, 20μL of acetic anhydride and a catalytic amount 4-dimethyl aminopyridinewere added. The mixture was left in a tightly closed vial for 4-16hours, and then it was extracted with 1 mL of aqueous HCl (1 N) and 1 mLof a Na₂CO₃ (1 N) solution. The products were analyzed by gaschromatography. A temperature gradient was used for elution. For thebenzyl derivative the conditions were as follows: 2 minutes at 175° C.,increase in temperature by 1.5° C./minute to 200° C., and then remainingfor 10 minutes at 200° C. For the isobutyl-substituted compound theconditions were as follows: 2 minutes at 165° C., and increase intemperature by 1.5° C./minute to 190° C. Baseline separation of all fourdiastereomers was achieved under these conditions and the product ratioswere calculated. For the 2-methyl substituted diester, KRED 1008produced a single diastereomer as 91% of the product mixture, KRED 1007produced a single diastereomer as 89% of the product mixture, KRED 1004produced a single diastereomer as 84% of the product mixture, and KRED1001 produced a single diastereomer as 65%. In the case of the 2-benzylsubstituted diester, KRED 1008 produced a single diastereomer as 100% ofthe product mixture; no other diastereomers were detected. In the caseof the 2-isobutyl substituted diester, KRED 1001 produced a singlediastereomer as 94% to 97% of the product mixture.

Example 7 Alternate Synthesis of diethyl 2-benzyl 3-ketoglutarate andReduction with 1008

In 60 mL of tetrahydrofuran (THF) 15 mL of 1,3 acetone dicarboxylate(0.072 mole) were dissolved. Under nitrogen atmosphere the mixture wascooled at —18° C. for 10 minutes before 74 mL (0.148 mole) of lithiumdiisopropylamine (LDA) solution (2M in hexane) was slowly added over aperiod of 20 minutes. After stirring the solution at —18° C. for 10minutes, 10 mL of tertahydrofuran mixed with 9.5 mL of benzyl bromide(0.080 mole) were slowly added, and the solution was stirred for another2.5 h (temperature was slowly increased to −10° C.) before completereaction was observed by thin layer chromatography analysis of reactionaliquots. The reaction mixture was then poured into 100 mL of an aqueoussolution containing 2M hydrochloric acid and 200 mL of ethyl acetate.The aqueous layer was extracted one more time with 100 mL of ethylacetate, and the combined organic layers were extracted once with 50 mLof brine. After drying of the organic layer with sodium sulfate, solventevaporation gave 25 g of an oily product that was incubated with enzyme1008 without any further purification according to the followingreaction conditions: In 250 mL of water containing 200 mM of potassiumphosphate (pH 6.8) 2.5 % (v/v) dimethyl sulfoxide, 0.2 M of sodiumchloride, 5 g of D-glucose, 0.1 g of NADP+ and 100 mg of lyophilizedglucose dehydrogenase and KRED 1008, 3.3 g of crude 2-benzyl3-ketoglutarate were added. The reaction was incubated in a shake ovenat 37° C. for 12 h before another batch of NADP+ (0.1 g), KRED 1008 (0.1g) and glucose dehydrogenase (0.1 g) were added to the reaction mixture.Incubation for 20 more hours gave complete conversion of the ketone tothe alcohol as shown by high pressure liquid chromatography and thinlayer chromatographic analysis of crude reaction extracts. The aqueousreaction mixture was then extracted with ethyl acetate (3×, 100 mL each)and the combined organic layers were washed with brine and dried withsodium sulfate. Solvent evaporation gave 3.2 g of an oily product fromwhich 2.2 g of pure diethyl 2-benzyl 3-hydroxy glutarate were isolatedafter silica gell chromatographic purification. Based on the recovery ofpure alcohol from the 3.3 g of crude diethyl 2-benzyl 3-ketoglutaratethe overall yield to 2-benzyl 3-hydroxy glutarate as calculated from thestarting 1,3 acetone dicarboxylate was 80%.

Example 8 Enzyme-catalyzed Diastereoselective Reduction of2-methyl-3-ketoglutarate diethyl ester

To 40 mL of 300 mM potassium phosphate buffer, pH=6.5, containing NaCl(100 mM), DMSO (3% v:v), glucose (200 mM), and glycerol (10% v:v), 40 mMof 2-methyl-3-ketoketoglutarate diethyl ester was added along with 100mg of lyophilized KRED 1008 and 30 mg of glucose dehydrogenase. Thereaction mixture was incubated for 48 hours at 37° C. Gaschromatographic analysis showed that the yield of hydroxy diesterproduct was greater than 80%. The product was isolated by extraction ofthe reaction mixture with ethyl acetate and purification using silicagel chromatography. Isolated yield was in the range of 85-95%.

Example 9 Enzyme-Catalyzed Diastereoselective Reduction of2-isobutyl-3-ketoglutarate diethyl ester

To 40 mL of 300 mM potassium phosphate buffer buffer, pH=6.5, containingNaCl (100 mM), DMSO (3% v:v), glucose (200 mM), and glycerol (10% v:v),40 mM of 2-isobutyl-3-ketoketoglutarate diethyl ester was added alongwith 100 mg of lyophilized KRED 1001 and 30 mg of glucose dehydrogenase.The reaction mixture was incubated for 48 hours at 37° C. Gaschromatographic analysis showed that the yield of hydroxy diesterproduct was greater than 80%. The product was isolated by extraction ofthe reaction mixture with ethyl acetate and purification using silicagel chromatography. Isolated yield was in the range of 85-95%.

Example 10 Reduction of 2-benzyl-3-ketoglutarate diethyl ester UsingWhole Cells Expressing KRED 1008

Whole cell reductions of 2-benzyl-3-ketoglutarate were carried out usingE. coli cells expressing the gene encoding KRED 1008. Cells were grownovernight at 30° C. in 400 mL of a Luria Broth/glucose (2g/L)/ampicillin (100 mg/L) media. The cells were isolated bycentrifugation and re-suspended in 400 mL of a minimal salt (M9)solution containing isopropylthiogalactoside (IPTG, 0.5 mM), glucose (5g/L), ampicillin (100 mg/L), and 2-benzyl-3-ketoglutarate diethyl ester.In independent experiments, the concentration of2-benzyl-3-ketoglutarate diethyl ester was varied over the range of 1gram/liter to 7.5 grams/liter. Cells continued to grow at 30° C. afteraddition of the ketodiester substrate. Samples were removed at regulartime intervals and analyzed for product formation using gaschromatography until complete reaction was observed. Although the2-benzyl-3-ketoglutarate diethyl ester substrate did not completelydissolve in the aqueous buffer, and although no DMSO co-solvent wasused, the reduction reactions were complete after 20-40 hours. Inreactions that were allowed to proceed for more that 24 hours, anotherportion of glucose (1-3 g) and IPTG (0.4 mL of a 0.5 M solution) wereadded. The product 2-benzyl-3-hydroxyglutarate diethyl ester wasisolated by removal of the cells by centrifugation and extraction of thereaction mixture with ethyl acetate. The yield of2-benzyl-3-hydroxyglutarate diethyl ester in all cases was greater than80%.

Example 11 Reduction of other 2-substituted-3-ketoglutarate diestersUsing Whole Cells Expressing Ketoreductase Genes

The procedure of Example 10 is repeated with E. coli cells expressing aketoreductase gene appropriate for the reduction of the target2-substituted-3-ketoglutarate diester. The product2-substituted-3-hydroxyglutarate diesters are isolated by removal of thecells and extraction of the reaction mixtures with ethyl acetate.

Example 12 Synthesis of 3-benzyloxaloacetate diethyl ester

In 7 mL tetrahydrofuran 1 g (4.8 milimole) diethyl oxaloacetate sodiumsalt was dissolved. In 5 mL tertahydrofuran 0.7 mL of benzyl bromide(5.9 milimole) are dissolved and added to the previous mixture. Thereaction was refluxed for 9 h before thin layer chromatography analysisshowed consumption of all reactants. Product purification started byadding the reaction mixture in 40 mL aqueous solution containing 0.5 MHCl and 40 mL ethyl acetate. The water layer was extracted one more timewith ethyl acetate and the combined organic solvents were back extractedwith brine, dryied with sodium sulfate and evaporated to dryness. Aftersilica gel chromatography 0.25 g (19% yield) of pure3-benzyloxaloacetate diethyl ester were isolated.

Example 13 Alternative Synthesis of 3-benzyloxaloacetate diethyl ester

1 mL (5.6 milimole) of ethyl hydroxyccinamate was dissolved in 10 mL oftetrahydrofuran, and the solution was cooled at −15° C. before 2.9 mL(7.3 milimole) of butyl lithium (2.5 M solution in hexanes) was slowlyadded. After stirring the reaction for 10 min at −15° C., 0.8 mL (8.4milimole) of diethyl oxalate were added, and the reaction was leftslowly to reach room temperature (about 2 hours). After stirring at roomtemperature overnight, the mixture was mixed with 70 mL of water(containing 0.1 M HCl) and extracted twice with diethyl ether. Combinedorganic extracts were back extracted with brine, dryed with sodiumsulfate and evaporated to dryness. Pure 3-benzyloxaloacetate diethylester (0.8 g, 51% yield) was obtained after silica gel chromatographicpurification of the crude oily reaction product.

Example 14 Enzyme-Catalyzed Diastereoselective Reduction of3-benzyloxaloacetate diethyl ester

The procedure of Example 4 was repeated for the screening of theKRED-8000 kit with 3-benzyloxaloacetate diethyl ester. Enzymes KRED 1008and KRED 1004 showed complete reduction to the alcohol. Both reactionswere repeated in larger scale for the purpose of isolating the productand verifying the structure using 1H NMR spectroscopy. In 20 mL of anaqueous solution containing 250 mM potassium phosphate (pH 6.7), 5%(v/v) polyethelene glycol (MW 1450) 0.5 M sodium chloride, 0.076 g ofNADP+, 3.5 g of D-glucose 0.1 g of both lyophilized enzymes glucosedehydrogenase and KRED 1004 or KRED 1008 0.5 g of 3-benzyloxaloacetatediethyl ester were added. The reactions were incubated at 37° C. in ashake oven for 20 h before they were extracted with ethyl acetate. Theorganic layers were evaporated to dryness, and the alcohols were furtherpurified using silica gel chromatography, before they were analyzed by¹H NMR. Both enzymes gave 2-hydroxy-3-benzylsuccinate diethyl ester asthe only product.

Example 15 Chiral Analysis of Enzymaticaly-Produced 3-Substitutedoxaloacetate diesters

For the determination of the stereochemistry of the ethyl 3-benzyl2-hydroxy succinate, standard compounds with known stereochemistry werechemically synthesized according to published literature procedure (Org.Synth, Vol 63, pg 109). The two enantiomeric ethyl 3-benzyl 2-hydroxysuccinates were separated using chiral HPLC chromatography (Column:CHRALCEL OD-RH, eluent: H₂O/MeOH, v/v, 70/30 flow rate: 1 mL/min).Analysis of the reaction product of diethyl-2-benzyl oxoglutarate andKRED 1008 showed only one peak that co-eluted with (2R, 3S) 3-benzyl2-hydroxysuccinate. KRED 1004, on the other hand, gave (2S, 3R) 3-benzyl2-hydroxysuccinate as the major product (62%) along with smaller amounts(14%) of the (2R, 3S) and another peak (24%) that is either the (2S, 3S)or (2R, 3R) diastereomer.

Example 16

Enzyme-catalyzed diastereoselective reduction of various2-substituted-3-ketoglutarate and 3-substituted oxaloacetate diesters.It is appreciated that using procedures analogous to those described inprevious Examples 1 to 15 the reduction of a wide range of differentvarious 2-substituted-3-ketoglutarate and 3-substituted oxaloacetatediesters can be produced. Using a screen such as that described inExample 4, the best enzyme for the diastereoselective reduction can beidentified, and the reaction can be carried out at a preparative scaleas described in Examples 6 to 11. Diastereoselective reduction of thefollowing representative compounds is envisioned:2-isobutyl-3-ketoglutarate dimethyl ester, 2-methyl-3-ketoglutaratedimethyl ester, 2-benzyl-3-ketoglutarate dimethyl ester,2-phenyl-3-ketoglutarate diethyl ester, 2-allyl-3-ketoglutarate diethylester, 2-propargyl-3-ketoglutarate diethyl ester,2-(4-pyridyl)-3-ketoglutarate diethyl ester, 2-isopropyl-3-ketoglutaratediethyl ester, 2-propyl-3-ketoglutarate diethyl ester,2-isopentyl-3-ketoglutarate diethyl ester, 2-(2-thienyl)-3-ketoglutaratediethyl ester, 3-isobutyloxaloacetate dimethyl ester,3-methyloxaloacetate dimethyl ester, 3-benzylyloxaloacetate dimethylester, 3-phenylyloxaloacetate diethyl ester, 3-isopentyloxaloacetatediethyl ester, 3-propyloxaloacetate diethyl ester, 3-allyloxaloacetatediethyl ester, 3-propargyloxaloacetate diethyl ester,3-(4-pyridyl)oxaloacetate diethyl ester, and 3-(2-thienyl)oxaloacetatediethyl ester.

Example 17 Screening to Find a Hydrolytic Enzyme Capable of Catalyzingthe Regioselective Hydrolysis of 2-benzyl 3-hydroxy-diethylketoglutarate

A Chirazyme Screening Set (BioCatalytics, Inc., Pasadena, Calif.) wasused to screen for a hydrolytic enzyme capable of catalyzing theregioselective hydrolysis of 2-benzyl-3-hydroxy-diethyl ketoglutarate,produced by stereoselective reduction as in Example 7. Each of the 11hydrolytic enzymes in the Chirazyme Screening Set (1 mg) was incubatedat 37° C. in a potassium phosphate buffer solution (250 mM, pH=7; 1 mLtotal volume) containing 40 mM of 2-benzyl 3-hydroxy-diethylketoglutarate and 5% (v/v) DMSO. The reaction progress was analyzed at 2h and 24 h using high performance liquid chromatography after eachsample was acidified to pH 1-2 by the addition of HCl and extracted withethyl acetate. Enzymes L2, L3, L8, L9 and El gave product. As shown inTable 1, all enzymes gave initially the mono-acid hydrolysis product of2-benzyl 3-hydroxy-diethyl ketoglutarate, which in most cases wasfurther hydrolyzed to produce the di-acid in significant yields, withthe exception of L9, which gave minor diacid product even after 24 h ofincubation with the substrate. Although the mono-acid product that wasobtained in all these reactions had the same retention time in HPLCanalysis, the reactions with L2, L3, L9 and E1 were scaled up (10 mLsolution, 80 μL substrate, 10 mg enzyme), and allowed to react for 4-10h until only the mono-acid was formed. Product was isolated afterextracting the reaction mixture with EtOAc at neutral pH to remove anyunreacted ester, followed by extraction at acidic pH, which removed allthe acids from the water. ¹H NMR analysis of all 4 reactions identifiedthe product to be the same less hindered mono-acid A (FIG. 6). Insupport of this conclusion, literature precedent predicts that the lesshindered ester group of 2-benzyl-3-hydroxy diethyl glutarate will reactmore rapidly in the presence of a hydrolytic enzyme (K Faber,Biotransformations in Organic Chemistry, 3^(rd) edition, Chapter 2,Springer Verlag, Berlin-Heidelberg-New York, 1997). Additional proof ofthe structure of the mono-acid was obtained after the L2- and chemical-(Example 18) hydrolyzed diethyl 2-benzyl 3-hydroxy glutarate wasrearranged with DPPA to the corresponding primary cyclic carbamate (FIG.7) and analyzed by ¹H NMR spectroscopy. Details for this reaction arepresented in Examples 24 and 25. TABLE 1 Enzymatic hydrolysis ofdiastereoselectively-reduced 2-benzyl-3- ketoglutarate diester. EnzymeTime (h) SM % Mono-acid Di-acid L2  2 h  38 62 — 24 h — 64 36 L3  2 h 70 30 — 24 h — 58 42 L8  2 h 100 — — 24 h  78 22 — L9  2 h  48 52 24 h— 91  9 E1  2 h  6 94 — 24 h — 50 50

Example 18 Regioselective Chemical Hydrolysis of 2-benzyl3-hydroxy-diethyl ketoglutarate

Mild chemical hydrolysis reaction conditions were tested for thehydrolysis of 2-benzyl-3-hydroxy diethyl glutarate coming from thereduction with KRED 1008 as in Example 7. In small-scale reactions (1.5mL total volume) various basic hydrolysis conditions (different solventsand ratios as well as NaOH concentrations) were prepared and thereaction progress was followed using HPLC analysis. Every sample thatwas analyzed was first acidified with glacial acetic acid and injectedwithout any other treatment onto a C18 reverse phase column. Table 2shows the two best reaction conditions found for the hydrolysis of2-benzyl-3-hydroxy diethyl glutarate. Incubating the hydroxydiester in amixture of Ethanol/H₂O (v/v, 2/8) containing differing amounts of NaOHand analyzing the reaction progress with HPLC showed the formation oftwo products. The compound with the longer retention time formedquickly, even under very mild hydrolysis conditions. Isolation and ¹HNMR analysis of this compound showed it to be a mono-ester, identical tothat obtained from the reactions with the regioselective hydrolysiscatalyzed by the hydrolytic enzyme in Example 17. TABLE 2 Hydrolysis ofKRED 1008-reduced 2-benzyl-3-ketoglutarate Conditions A^(a) ConditionsB^(b) Time SM Mono-acid Di-acid SM Mono-acid Di-acid 0.0^(c) h 13% 87%87% 13% 0.5 h 75% 25% 8% 92% 1.5 h 52% 48%^(a)Conditions A: H₂O/EtOH (v/v, 8/2) 0.05M substrate, 0.25M NaOHincubate at RT.^(b)Conditions B: H₂O/EtOH (v/v, 8/2) 0.05M substrate, 0.075M NaOHincubate at RT.^(c)This time point was taken immediately after the mixing of substrateand the NaOH solution.

The possibility of isomerization of either of the two chiral centers ofthe alcohols under the hydrolysis conditions was then evaluated. Themonoacid product of the chemical hydrolysis under Conditions B wasisolated, esterified with CH₂N₂, and analyzed by chiral gaschromatography. No new peaks were detected, thus indicating that noisomerization of either chiral center occurred under the chemicalhydrolysis conditions employed.

Example 19 Whole Cell Reduction of 2-methyl 3-hydroxy diethyl glutarateand enzymatic hydrolysis

After establishing that some of the lipases were able to hydrolyze theKRED 1008-reduced 2-benzyl 3-hydroxy diethyl glutarate, the same set ofenzymes was tested for its ability to hydrolyze the KRED 1008-reduced2-methyl 3-hydroxy diethyl glutarate. This compound was obtained afterthe whole cell reaction of 2-methyl diethyl 3-ketoglutarate usingrecombinant E. coli expressing KRED 1008 as described in Example 10. Amixture of diastereomers was obtained, and because the identify of theabsolute stereochemistry of each one is currently unknown, they areindicated as A, B, C, and D in Table 3. From the retention times of eachcompound in the chiral GC, two sets of enantiomeric pairs wereidentified. Peaks A and B were assigned as representing on enantiomericpair and peaks C and D were assigned as representing the otherenantiomeric pair. Interestingly, the yield of the major diastereomer(indicated as C) in the whole-cell reactions dropped to ˜75% from the91% (Example 6) yield that was obtained in the cell-free reductionsusing lyophilized 1008. This was most likely due to the competingreductions of the starting material by native E. coli ketoreductases.TABLE 3 Hydrolysis of KRED 1008-reduced 2-methyl 3-hydroxy diethylglutarate Starting material Product Yield Isomers Yield Isomers Enzyme %(%) % (%) L2 0 A&B (0) >98 A(17) B(6) C&D (0) C(66) D(11) L3 10 A&B (50)90 A(12) B(6) C&D (50) C(77) D(5) L5 80 A&B (˜0) 20 A(24) B(16) C(>98)D(˜0) C(18) D(42) E1 0 A&B (0) >98 A(15) B(6) C&D (0) C(75) D(4)

Enzymatic hydrolysis reactions were performed as follows: Solutions (2m]L) containing 300 mM potassium phosphate (pH=7), 2 mg enzyme and 30 mMof substrate were incubated at 37° C. in a shake oven. As shown in Table3 above, out of all 9 enzymes only L2, L3, L5 and El showed good productformation after 1.5 h of reaction at 37° C. Product identification wasachieved as follows: after 1.5 h of incubation at 37° C. each reactionwas acidified to pH-1-2 with HCl (1N) addition and the solutions wereextracted twice with ethyl acetate. The combined organic layers wereevaporated to dryness, and the oily product was re-dissolved in diethylether and treated with an excess of CH₂N₂ before it was analyzed usingchiral GC chromatography. Under the conditions employed for analysis,all four diastereomers of the 2-methyl 3-hydroxy methyl ethyl glutarate(coming from hydrolysis and reaction with CH₂N₂) and the fourdiastereomers of the starting material, 2-methyl 3-hydroxy diethylglutarate, were clearly separable. Integration of these product mixturesgave the numbers presented in Table 3 above. Notice the reaction with L5where the enzyme reacted more slowly with the major diastereomer, givingafter 1.5 h of reaction a single diastereomer in as unreacted startingmaterial in high yields. This example shows how the stereoselectivity ofthe hydrolytic enzyme can be a useful adjunct to the diastereoselectivereduction. By selecting a hydrolytic enzyme (in this case, L5) thatcatalyzes the hydrolysis of the diastereomeric diesters at differentrates, the diastereoselectivity of the monoacid or diester obtained canbe improved. In this case, the diastereomer C of the diester is obtainedas the only diastereomer after hydrolysis of the minor contaminatingdiastereomeric diesters.

Example 20 Chemical and Enzymatic Hydrolysis of 2-isobutyl 3-hydroxydiethyl glutarate

The mild hydrolysis conditions that were shown to give a singlemono-ester product with 2-benzyl 3-hydroxy diethyl glutarate wereutilized for the hydrolysis of the KRED 1001-reduced 2-isobutyl3-hydroxy diethyl glutarate. Under these conditions, in 4 mL of H₂O/EtOH(8:2, v:v) containing 50 mM of NaOH, 25 mg (25 mM) of 2-isobutyl3-hydroxy diethyl glutarate were added and the reaction was stirred for1.5 h at room temperature. Product purification was achieved afteracidification of the reaction mixture with HCl and extraction withEtOAc. Proton NMR analysis of the crude isolated product revealed theformation of a single mono-ester product, which was the outcome of thehydrolysis of the less hindered ester group A (FIG. 6).

Enzymatic methods using the previously utilized hydrolytic enzymes werealso tested for hydrolysis of 2-isobutyl 3-hydroxy diethyl glutarate.Under these reaction conditions, 2 mL of a solution containing potassiumphosphate buffer 300 mM (pH=7), 5% v/v DMSO, 2 mg of each enzyme and 10μL of 2-isobutyl 3-hydroxy diethyl glutarate were incubated at 37° C. ina shake oven. The reaction progress was monitored as follows: atspecific time points samples (0.3 mL) from each reaction mixture weretaken, acidified to pH −2 with HCl (2N), extract with ethyl acetate (0.3mL) and dried with MgSO₄. The organic layer was then evaporated todryness, mixed with 0.4 mL of Et₂O/CH₂N₂ mixture to form the methylester and analyzed with GC chromatography (Chiral column, isocratic 170°C.). All three diethyl, mono-ethyl-mono-methyl and di-methyl productswere easily separated. Proof of the structure of the mono-acid wasobtained after the L2-hydrolyzed diethyl 2-isobutyl 3-hydroxy glutaratewas rearranged with DPPA to the corresponding primary cyclic carbamate(FIG. 7) and analyzed by ¹H NMR spectroscopy. Details for this reactionare presented in Examples 24 and 25. TABLE 4 Enzymatic hydrolysis using2-isobutyl 3-hydroxy diethyl glutarate Time Mono acid Di-acid SM Enzyme(h) Yield A/B^(a) Yield A/B Yield A/B^(a) L2 2 h 29% 97/3 71% 94/6 24 h100% 96/4 L3 2 h 100% 94/6 24 h 100% 94/6 L9 2 h 24 h 100% 95/5 E1 2 h100% 96/4 24 h 86% 93/7 14% 100/0  32 h 47% 90/10 53% 97/3  E2 2 h 100%96/4 24 h 91% 96/4 9% 89/11 32 h 88% 95/5 12% 89/11^(a)Ratio of diastereomers.

As described in Example 6, two diastereomers (indicated as A and B inthe above Table 4) in a ratio of 97% to 3% were formed from theenzymatic reduction of 2-isobutyl 3-keto diethyl glutarate and KRED1001. As a result, the stereoselectivity of the enzymatic hydrolysis wasalso investigated. The previous methylated samples were evaporated todryness and treated with a solution (0.3 mL) of CH₂Cl₂ that contained anexcess of Ac₂O and catalytic a amount of TMSOTf. Under these conditionsthe alcohols are acetylated and the diastereomers separate in Chiral GC(retention times: 19.1 min di-ethyl, 15.2 min mono-ethyl-mono-methyl;12.5 min di-methyl; Chiral column; 145° C. for 2 min then 145 to 180° C.at 0.5° C./min). The ratio of diastereomers that was forming during theenzymatic hydrolysis is shown in the Table 4 above. The mono-acid thatis formed in each reaction is the same and corresponds to the lesshindered mono-acid A (FIG. 6). In the case of enzymes E1 and E2 furtherhydrolysis of the more hindered ester to the diacid was obtained whenthe reactions were incubated for longer times. In the case of El, thetwo diastereomers were hydrolyzed at slightly different rates. Thisexample further illustrates how stereoselective hydrolysis cataluzed byan enzyme can further improve the stereoisomeric purity of the mono-acidor diacid products.

Example 21 Chemical and Enzymatic Hydrolysis of3-benzyl-2-hydroxysuccinate diethyl ester

Both mild chemical and enzymatic conditions were tested for thehydrolysis of 3-benzyl 2-hydroxysuccinate. (2S, 3R) 3-Benzyl2-hydroxysuccinate (150 μL, 0.53 mmole) was incubated with a mixture of2 mL of an aqueous NaOH (0.5 M) solution and 0.4 mL of ethanol. Afterstirring for 2 h at RT, acidification and extraction of the reactionmixture with EtOAc gave a single product, which was identified as eitherthe mono-acid A or B (FIG. 6) using ¹H NMR spectroscopy. TABLE 5Enzymatic hydrolysis of 3-benzyl 2-hydroxysuccinates Time Mono-acidStarting material Enzyme Substrate (h) Yield (%) A/B^(a) Yield (%)A/B^(a) L6 2S, 3R 4 h 11 100/0 89 90/10 24 h 46 100/0 54 85/15 2R, 3S 4h 0 100 87/13 24 h 7 92 87/13 L7 2S, 3R 4 h 12 88 90/10 24 h 53 100/0 4782/17 2R, 3S 4 h 7 93 86/14 24 h 30  81/19 68 85/15^(a)Ratio of diastereomers in reactant and product.

Enzymatic hydrolysis of both the (2S, 3R) and (2R, 3S) 3-benzyl2-hydroxysuccinates was then investigated using the Chirazyme screeningkit. Both these compounds were synthesized chemically according toliterature procedures (Org. Synth. Vol 63, pg. 109) and both containedabout 10-13% of a diastereomeric impurity, which was the (2S, 3S) forthe synthesis of (2S, 3R) and the (2R, 3R) for the synthesis of (2R,3S). Aqueous solutions (2 mL) containing 250 mM Kpi pH 7, 5% v/v DMSO 5μL/mL of each enantiomer and 2 mg/mL of each lipase were incubated in ashake oven at 37° C. Samples were taken and analyzed using BPLCchromatography. Both enantiomers after 24 h of incubation werehydrolyzed to the mono-acid by lipase L2, L7 and E1, while L6 seems toreact faster with the (2S, 3R) enantiomer. All hydrolysis reactions gavea single product, which had the same retention time (at HPLC analysis)with the ¹H NMR-characterized product of the mild chemical hydrolysis.In a second experiment, hydrolysis reactions of (2S, 3R) and (2R, 3S)3-benzyl 2-hydroxysuccinates using L6 and L7 under identical conditions(Kpi, 250 mM pH 7, 5% v/v DMSO, 2 mg/mL enzyme and 5 μL/mL diester) wereperformed and the reaction progress as well as the ratio ofdiastereomers in both the starting materials and the products weremeasured. As shown in Table 5 above, L6 hydrolyzed preferably the (2S,3R) 3-benzyl 2-hydroxysuccinate isomer. The same selectivity for thehydrolysis of the (2S, 3R) isomer was also observed in the hydrolysisusing L7. However, this enzyme also reacted with lower rates with theother diastereomers of 3-benzyl 2-hydroxysuccinate.

Example 22 Formation of the More Hindered Mono-Acid of 2-benzyl3-hydroxy glutarate

Formation of the more hindered mono-acid B provides a useful precursorfor rearrangement. (FIG. 6). This molecule was synthesized in a two-stephydrolysis-esterification process. Since the less hindered mono-acid Awas preferably forming under mild hydrolysis conditions as well as inthe reactions with certain commercially-available lipases, it washypothesized that it would also be esterified much faster if the2-benzyl 3-hydroxy glutarate diacid reacts with ethanol under mildconditions. As a result, incubating 2.1 grams (7.2 mmole) of 2-benzyl3-hydroxy diethyl glutarate in 7 mL of a 5 M (35 mmole) KOH aqeuoussolution also containing 1 mL EtOH at room temperature gave completeconversion to the diacid (I) (FIG. 7) after stirring for 3-4 h. Theproduct was isolated (1.6 gr, 95% yield) after acidification of thereaction mixture with HCl and extraction with EtOAc. Selectiveesterification was achieved when diacid (I) was stirred in 25 mL of EtOHat 45° C. for 18 hours in the presence of one drop of concentratedH₂SO₄. The reaction was followed by HPLC and only the mono-ester II(FIG. 7) was shown to form under the reaction conditions. After thereaction was complete, ethanol was evaporated to ˜1-2 mL 30 mL EtOAcwere added and extracted once with 5 mL of water (0.05N HCl). Solventevaporation gave 1.6 grams (Yield 90%) of mono-acid II (FIG. 8).

In a different method the first extraction was eliminated. According tothis method, 4.5 g (0.015 mole) of diethyl 2-benzyl-3-hydroxyethyl-glutarate are dissolved in 3 mL ethanol and were mixed with 15 mLof water containing 1.8 g (0.045 mole) of sodium hydroxide. Aftervigorous stirring at room temperature for 3 h HPLC analysis of a crudereaction mixture showed complete formation of the diacid I (FIG. 8). Atthis point the mixture was acidified to pH˜2-3 with addition of 6M HClsolution and the solvent was evaporated to dryness under reducedpressure. The precipitate redissolved in 80 mL of ethanol, and, afterfiltering to remove the insoluble sodium chloride salts, it wasconcentrated under reduced pressure to 40 mL to azeotrope any remainingwater from the mixture. A drop of concentrated HCl was then added, andthe homogeneous mixture was stirred at 40° C. for 14 h, where completeconversion to the more hindered mono acid II (FIG. 8) was obtained.Sometimes if long reaction times were allowed, 2-5% of the diester alsoformed. Solvent evaporation of the reaction mixture to dryness gave 3.9g of the mono-acid product (yield 98%).

Example 23 Formation of the More Hindered mono-acid of diethyl2-isobutyl 2-hydroxy ketoglutarate

The first procedure of Example 22 was repeated except that diethyl2-benzyl 2-hydroxy ketoglutarate was replaced with 2-isobutyl 2-hydroxyketoglutarate. The product was the more hindered mono-acid that wasisolated in 84% yield. It will be appreciated that the general procedurecan be used in the practice of the present invention by one skilled inthe art to produce a wide range of different more hindered mono-acidproducts.

Example 24 Rearrangement of mono-ethyl 2-benzyl 3-hydroxy glutarate (II,FIG. 8) with diphenylphosphoryl azide to the cyclic carbamate

In 30 mL of toluene, 2 g (7.6 milimole) of the KRED-1008-producedmono-ethyl 2-benzyl 2-hydroxy ketoglutarate (II, FIG. 8) were dissolvedalong with 2 μL (9.3 milimole) and 0.8 (7.8 milimole) of triethylamine.The reaction was heated at 80° C. and intense bubbling was observed.After 2.5 h at 80° C. the temperature was decreased to 60° C. and wasleft stirring for another 4 to 8 h. At the end of the reaction 30 mL ofethyl acetate were added to the reaction mixture, and was extracted oncewith 20 mL (0.25 N HCl) followed by extraction with 20 mL of saturatedmono sodium carbonate (NaHCO₃). The organic layer was then washed withbrine and evaporated to dryness giving 2 g of an oily precipitate. Purecyclic carbamate (FIG. 7) was obtained (1.2 g, 4.6 milimole) aftersilica gel purification in 60% isolated yield. Proton and carbon NMR aswell as MS analysis all confirmed the correct structure. In additioncareful proton decoupling and NOE experiments of the hydrogens presenton the carbamate ring showed that their relative position is cis. Basedon this result, the single diastereomer that was obtained in thisreaction sequence using KRED 1008 can be assigned as having either the(3R, 4S) or (3S, 4R) absolute stereochemistry.

Example 25 Rearrangement of mono-ethyl 2-isobutyl 3-hydroxy glutarate(II, FIG. 8) with diphenylphosphoryl azide to the cyclic carbamate

The procedure of Example 24was repeated except that mono-ethyl 2-benzyl3-hydroxy glutarate was replaced with mono-ethyl 2-isobutyl 3-hydroxyglutarate. The latter was synthesized from diethyl 2-isobutylketoglutarate after reduction with KRED 1001. As described above, protonNMR decoupling and NOE experiments showed that the hydrogens on thecarbamate ring possessed the cis relative orientation, which isconsistent with either (3R, 4S) or (3S, 4R) absolute stereochemistries.Thus, the single diastereomer obtained in this reaction sequence usingKRED 1001 had the either (3R, 4S) or (3S, 4R) absolute stereochemistry.

Example 26 Alternative Rearrangement Conditions formono-ethyl-2-benzyl-3-hydroxy glutarate

Synthesis of the amine was also achieved under Hofmann rearrangementconditions starting from the corresponding amide. In 20 mL of methelenechloride containing a drop of dimethyl formamide, 1 g (3.8 millimole) ofthe hindered mono-acid of 2-benzyl 3-hydroxy glutarate (II, FIG. 8) wasdissolved. After addition of 0.45 mL (5.1 millimole) of oxallylchloride, the reaction was stirred at room temperature for 1 hour beforeammonia gas started bubbling through a stainless steel needle immersedin the reaction solution. After one hour of bubbling, the solution wasfiltered and evaporated to dryness. Silica gel purification of the oilyproduct obtained after solvent evaporation gave 0.5 g (50% yield) of theamide. Rearrangement of the amide was performed when 0.16 g of (0.6millimole) were dissolved in 0.4 mL of acetonitrile and added to 0.4 mLof an aqueous solution containing 0.4 g (0.9 millimole) of[bis(trifluoroacetoxy)iodo]benzene [(CF₃CO₂)₂PhI]. After stirring themixture for 4 hours at room temperature, 0.6 mL of a NaHCO₃ saturatedsolution and 15 mL EtOAc are added. The mixture was filtered to removethe insoluble materials, and the two layers were allowed to separate.After isolation of the organic layer, drying with Na₂SO₄ and evaporationto dryness, 0.1 g (62% yield) of a cyclic carbamate as described inExamples 24 and 25 was obtained. The yields of amide formation can beimproved by using a different method employing first reaction of thefree acid with ethyl chloroformate followed by reaction with ammonia(Organic Syntheses CV8, 132). This reaction can be performed tosynthesize the amide of the protected alcohol (synthesized as describedin Examples 24 and 25) and then rearranged to the free amine with[bis(trifluoroacetoxy)iodo]benzene.

Example 27 Protection of the Hydroxy Group of mono-ethyl 2-benzyl3-hydroxy glutarate (II, FIG. 8) as acetate

After checking various methods for the acetylation of the alcohol in thepresence of the free carboxyl group, it was identified that catalyticamounts of TMSOTf (trifluoromethanesulfonic acid trimethylsillilester)in the presence of acetic anhydride gave quantitative yields of theacetyl-protected alcohol. In a typical protocol, 0.5 g (1.9 milimole) ofmono-ethyl 2-benzyl 3-hydroxy glutarate and 0.28 mL (2 milimole) ofacetic anhydride and 5 μL of TMSOTf were dissolved in 10 m]L ofmethylene chloride. The reaction was stirred at 4° C. for 15 to 30minutes before TLC analysis showed complete consumption of startingmaterial. Evaporation of solvent and silica gel purification gave 0.5 g(5 milimole, 87% yield) of pure acetylated product.

Example 28 Protection of the Hydroxy Group of mono-ethyl 2-benzyl3-hydroxy glutarate (II, FIG. 8) as tert-butyl dimethylsillyl (TBDMS)

In 5 mL of acetonitrile, 0.6 g (2.3 milimole) of mono-ethyl 2-benzyl3-hydroxy glutarate and 0.6 g (4 milimole) tert-butyldimethylsillylchloride (TBDMSCI) were dissolved. The mixture was cooledat −18° C. for 10 minutes before 0.8 mL (5.2 milimole) of 1.8diazabicyclo[5.4.0]undec-7-ene (DBU) were added. The reaction mixturewas left to warm to 4° C. in 2 hours and was stirred overnight at thistemperature. At the end of the reaction 30 mL of ethyl acetate wereadded to the reaction mixture and extracted with 10 mL of 0.2 M HClaqueous solution. Organic layer was dried with sodium sulfate, andevaporated to dryness. Pure protected alcohol (0.6 g, 69% yield) wasisolated after silica gel chromatographic purification. It is importantto note that if the reaction was not performed at low temperature, adifferent product (probably the protected carboxylic acid) waspredominantly forming. A small amount of this impurity also formed underthe aforementioned low temperature reaction conditions.

Example 29 Rearrangement of the TBDMS-Protected mono-ethyl 2-benzyl3-hydroxy glutarate with diphenylphosphorylazide

In 5 mL toluene 0.50 g (1.3 milimole) of TBDMS-protected mono-ethyl2-benzyl 3-hydroxy glutarate along with 0.34 mL (1.6 milimole)diphenylphosphorylazide (DPPA) and 0.15 mL (1.44 milimole) oftriethylamine (TEA) were dissolved. The reaction was heated at 75° C.for 2 h and then it was allowed to cool at 60° C. and stirred overnight.The next day, 4 mL of HCl (3M) were added to the mixture under vigorousstirring. The reaction was stirred for 30 minutes before 30 mL of ethylacetate were added. The organic layer was removed and extracted oncewith 10 mL of a saturated solution of NaHCO₃. After washing with brineand drying with NaSO₄ the solvent was evaporated and an oily product wasrecovered. Silica gel purification gave 0.4 g of a compound thatappeared to be (by proton NMR) the free amine. Mass spectrum analysisshowed a parent ion peak (MW+1) of 324, which was consistent with thefree acid and amine of the TBDMS-protected alcohol. Both the two aqueoussolutions (HCl and NaHCO₃) that were recovered from the extraction ofthe organic layer were analyzed to identify if hydrolyzed amino acidproduct was present. Nothing was detected besides DPPA byproducts in theNaHCO₃ layer.

It will be appreciated that the methods described herein provide asynthetic route to a number of novel, hydroxy-amino esters having thegeneral formulaH₂N—CHR¹—CH(OH)—(CH₂)_(n)—COOR³wherein (a) n is 0; R¹ is selected from the group consisting of alkyl,substituted alkyl, unsaturated alkyl, aryl, substituted aryl,heterocyclic, and substituted heterocyclic; and R³ is selected from thegroup consisting of alkyl; substituted alkyl, unsaturated alkyl, aryl,substituted aryl, aralkyl, substituted aralkyl, heterocyclic, andsubstituted heterocyclic, or (b) n is 1; R¹ is selected from the groupconsisting of methyl, ethyl, propyl, isopropyl, substituted alkyl,unsaturated alkyl, aryl, substituted aryl, heterocyclic, and substitutedheterocyclic; and R³ is selected from the group consisting ofsubstituted alkyl, unsaturated alkyl, aryl, substituted aryl, aralkyl,substituted aralkyl, heterocyclic, and substituted heterocyclic.

More particularly, if one starts with the 2-substituted-3-ketoglutarateor 3-substituted oxaloacetate diesters listed in Example 16, theinvention provides a number of hydroxy-amino esters, which aresummarized according to their respective starting diesters and R groupsin the following table: Hydroxy-amino ester Starting diester n R1 R32-phenyl-3-ketoglutarate diethyl ester 1 phenyl ethyl2-allyl-3-ketoglutarate diethyl ester 1 allyl ethyl2-propargyl-3-ketoglutarate diethyl ester 1 propargyl ethyl2-(4-pyridyl)-3-ketoglutarate diethyl ester 1 pyridyl ethyl2-isopropyl-3-ketoglutarate diethyl ester 1 isopropyl ethyl2-propyl-3-ketoglutarate diethyl ester 1 propyl ethyl2-(2-thienyl)-3-ketoglutarate diethyl ester 1 thienyl ethyl3-isobutyloxaloacetate dimethyl ester 0 isobutyl methyl3-methyloxaloacetate dimethyl ester 0 methyl methyl3-benzylyloxaloacetate dimethyl ester 0 benzyl methyl3-phenylyloxaloacetate diethyl ester 0 phenyl ethyl3-isopentyloxaloacetate diethyl ester 0 isopentyl ethyl3-propyloxaloacetate diethyl ester 0 propyl ethyl 3-allyloxaloacetatediethyl ester 0 allyl ethyl 3-propargyloxaloacetate diethyl ester 0propargyl ethyl 3-(4-pyridyl)oxaloacetate diethyl ester 0 pyridyl ethyl3-(2-thienyl)oxaloacetate diethyl ester 0 thienyl ethyl.

1. A method for producing a hydroxy-amino ester, comprising: contactinga substituted β-ketodiester having a ketone group and two esterfunctional groups with a ketoreductase under conditions permitting thereduction of the ketone group to a hydroxyl group; regioselectivelyhydrolyzing one of the ester functional groups to the correspondingcarboxylic acid, whereby a non-hydrolyzed ester functional groupremains; and converting the carboxylic acid group to an amine to producea hydroxy-amino ester.
 2. The method of claim 1, wherein theβ-ketodiester is a 3-substituted-oxaloacetic diester.
 3. The method ofclaim 1, wherein the β-ketodiester is a 2-substituted-3-ketoglutaratediester.
 4. The method of claim 1, wherein the β-ketodiester is acompound having the following formula:R²OOC—CHR¹—CO—(CH₂)_(n)—COOR³ wherein: n is 0 or 1, and R¹, R², and R³are each independently selected from the group consisting of alkyl,substituted alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl,heterocyclic, and substituted heterocyclic.
 5. The method of claim 4,wherein n is
 0. 6. The method of claim 5, wherein n is
 1. 7. The methodof claim 1, wherein the carboxylic acid is converted to an amine by theCurtius rearrangement.
 8. The method of claim 7, wherein the Curtiusrearrangement is carried out using the reagent diphenylphosphoryl azide.9. The method of claim 7, wherein the Curtius rearrangement is carriedout by converting the carboxylic acid to the corresponding acyl chlorideand then reacting the acyl chloride with an azide salt.
 10. The methodof claim 9, wherein the conversion of the carboxylic acid to thecorresponding acyl chloride is carried out by reacting the carboxylicacid with oxalyl chloride.
 11. The method of claim 4, wherein the esterfunctional group containing R² is hydrolyzed to the correspondingcarboxylic acid.
 12. The method of claim 4, wherein R¹ is selected fromthe group consisting of alkyl and phenyl.
 13. The method of claim 1,wherein the reduction of the ketone is substantially diastereoselective.14. The method of claim 1, wherein the hydroxyl group is protected priorto the conversion of the carboxylic acid to an amine .
 15. The method ofclaim 1, wherein the regioselective hydrolysis of only one of the esterfunctional groups to the corresponding carboxylic acid is catalyzed byan enzyme.
 16. The method of claim 1, wherein at least 90% of the oneester functional group is hydrolyzed to the corresponding carboxylicacid.
 17. The method of claim 1, wherein at least 95% of the one esterfunctional group is hydrolyzed to the corresponding carboxylic acid. 18.A compound having the formula:H₂N—CHR¹—CH(OH)—(CH₂)_(n)—COOR³ wherein (a) n is 0; R¹ is selected fromthe group consisting of alkyl, substituted alkyl, unsaturated alkyl,aryl, substituted aryl, heterocyclic, and substituted heterocyclic; andR³ is selected from the group consisting of alkyl; substituted alkyl,unsaturated alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl,heterocyclic, and substituted heterocyclic; or (b) n is 1; R¹ isselected from the group consisting of methyl, ethyl, propyl, isopropyl,substituted alkyl, unsaturated alkyl, aryl, substituted aryl,heterocyclic, and substituted heterocyclic; and R³ is selected from thegroup consisting of substituted alkyl, unsaturated alkyl, aryl,substituted aryl, aralkyl, substituted aralkyl, heterocyclic, andsubstituted heterocyclic.
 19. The compound of claim 18, wherein n is 0.20. The compound of claim 19, wherein R¹ is isobutyl, methyl, or benzyl;and R³ is methyl.
 21. The compound of claim 19, wherein R¹ is phenyl,isopentyl, propyl, allyl, propargyl, 4-pyridyl, or thienyl; and R³ isethyl.
 22. The compound of claim 18, wherein n is
 1. 23. The compound ofclaim 22, wherein R¹ is phenyl, allyl, propargyl, 4-pyridyl, isopropyl,propyl, or 2-thienyl; and R³ is ethyl.